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J. Biol. Chem., Vol. 281, Issue 22, 15412-15422, June 2, 2006

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The Inhibitory Subunit of the Rod cGMP Phosphodiesterase Binds the Catalytic Subunits in an Extended Linear Structure^*

Lian-Wang Guo¹, Hakim Muradov, Abdol R. Hajipour^¶, Michael K. Sievert, Nikolai O. Artemyev, and Arnold E. Ruoho

From the Department of Pharmacology, University of Wisconsin Medical School, Madison, Wisconsin 53706, the Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, and the ^¶Pharmaceutical Laboratory, College of Chemistry, Isfahan University of Technology, Isfahan 84156, Iran

The unique feature of rod photoreceptor cGMP phosphodiesterase (PDE6) is the presence of inhibitory subunits (P), which interact with the catalytic heterodimer (P) to regulate its activity. This uniqueness results in an extremely high sensitivity and sophisticated modulations of rod visual signaling where the P/P interactions play a critical role. The quaternary organization of the heterotetramer is poorly understood and contradictory patterns of interaction have been previously suggested. Here we provide evidence that supports a specific interaction, by systematically and differentially analyzing the P-binding regions on P and P through photolabel transfer from various P positions throughout the entire molecule. The P N-terminal Val¹⁶–Phe³⁰ region was found to interact with the P GAFa domain, whereas its C terminus (Phe⁷³–Ile⁸⁷) interacted with the P catalytic domain. The interactions of P with these two domains were bridged by its central Ser⁴⁰–Phe⁵⁰ region through interactions with GAFb and the linker between GAFb and the catalytic domain, indicating a linear and extended interaction between P and P. Furthermore, a photocross-linked product () was specifically generated by the double derivatized P, in which one photoprobe was located in the polycationic region and the other in the C terminus. Taken together the evidence supports the conclusion that each P molecule binds P in an extended linear interaction and may even interact with both P and P simultaneously.

Received for publication, January 20, 2006 , and in revised form, March 30, 2006.

^* This work was supported by National Institutes of Health Grants GM33138 (to A. E. R.) and EY10843 (to N. O. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables SI and SII.

¹ To whom correspondence should be addressed: 1300 University Ave., Madison, WI 53706. Tel.: 608-263-3980; Fax: 608-262-1257; E-mail: lianwangguo{at}wisc.edu.

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